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Chromosome analysis and sorting

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DOI: 10.1002/cyto.a.24324

REVIEW ARTICLE

Chromosome analysis and sorting

Jaroslav Doležel1 | Sergio Lucretti2 | István Molnár1 | Petr Cápal1 |

Debora Giorgi2

1
Institute of Experimental Botany of the Czech
Academy of Sciences, Centre of the Region Abstract
Haná for Biotechnological and Agricultural Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered
Research, Olomouc, Czech Republic
2 by only a few laboratories worldwide. Yet, it has been contributing significantly to pro-
Italian National Agency for New
Technologies, Energy and Sustainable gress in plant genetics, including the production of genome assemblies and the cloning of
Economic Development (ENEA), Division of
important genes. The dissection of complex genomes by flow sorting into the individual
Biotechnology and Agroindustry, Rome, Italy
chromosomes that represent small parts of the genome reduces DNA sample complexity
Correspondence
and streamlines projects relying on molecular and genomic techniques. Whereas flow
Jaroslav Doležel, Institute of Experimental
Botany of the Czech Academy of Sciences, cytometric analysis, that is, chromosome classification according to fluorescence and light
Centre of the Region Haná for Biotechnological
scatter properties, is an integral part of any chromosome sorting project, it has rarely
and Agricultural Research, Šlechtitelů 31,
CZ-779 00 Olomouc, Czech Republic. been used on its own due to lower resolution and sensitivity as compared to other cyto-
Email: dolezel@ueb.cas.cz
genetic methods. To perform chromosome analysis and sorting, commercially available
Funding information electrostatic droplet sorters are suitable. However, in order to resolve and purify chromo-
The European Regional Development Fund,
somes of interest the instrument must offer high resolution of optical signals as well as
Grant/Award Number: Plants as a Tool for
Sustainable Global Development; The Italian stability during long runs. The challenge is thus not the instrumentation, but the adequate
Ministry of Agriculture, Grant/Award Number:
sample preparation. The sample must be a suspension of intact mitotic metaphase chro-
Progetto MAPPA 5A (D.M. 7398/7303/08);
European Regional Development Fund, Grant/ mosomes and the protocol, which includes the induction of cell cycle synchrony, accumu-
Award Number:
lation of dividing cells at metaphase, and release of undamaged chromosomes, is time
CZ.02.1.01/0.0/0.0/16_019/0000827
consuming and laborious and needs to be performed very carefully. Moreover, in addi-
tion to fluorescent staining chromosomal DNA, the protocol may include specific labelling
of DNA repeats to facilitate discrimination of particular chromosomes. This review intro-
duces the applications of chromosome sorting in plants, and discusses in detail sample
preparation, chromosome analysis and sorting to achieve the highest purity in flow-
sorted fractions, and their suitability for downstream applications.

KEYWORDS
cell cycle synchronization, mitotic metaphase chromosomes, liquid chromosome suspension,
DNA isolation, DNA amplification, gene mapping and cloning, genome sequencing, marker
development, repetitive DNA labelling

1 | I N T RO DU CT I O N transmitting hereditary information and in generating genetic varia-

tion. The most frequent method to study chromosome organization
Studies initiated during the last quarter of the nineteenth century and behavior had been optical microscopy. However, in the last quar-
gradually revealed the key roles of chromosomes in storing and ter of the twentieth century, efforts were made to employ flow

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited and is not used for commercial purposes.
© 2021 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Cytometry. 2021;1–15. wileyonlinelibrary.com/journal/cytoa 1

2 DOLEŽEL ET AL.
cytometry for fast and quantitative characterization of the human
chromosome complement (flow karyotyping) to replace laborious
microscopic observation [1, 2]. Unfortunately, these expectations
were not fulfilled. Instead, molecular cytogenetics was established
and provided much higher resolution and more detailed information
concerning the molecular organization of human, animal and plant
chromosomes. Although flow karyotyping cannot compete with
molecular cytogenetics, it is fair to point to a few exceptions, such as
in the analysis of wheat cultivars where it detected recombined chro-
mosomes whose presence was previously unknown [3].
The availability of methods for flow cytometric chromosome anal-
ysis also opened avenues for purification of specific chromosomes by
flow sorting. Interestingly, this method contributed to the develop-
ment of chromosome painting [4, 5] that revolutionized mammalian
cytogenetics. Chromosome sorting also played an important role at
the beginning of the human genome sequencing project through facil-
itating the mapping of genes to chromosomes and the development
of chromosome-specific DNA libraries [6, 7]. In plants, chromosome
sorting has been employed in many applications, ranging from the
targeted development of DNA markers [8–11], the construction of
DNA libraries [12, 13], gene cloning [14, 15], genome sequencing
[16–18], and the validation of whole genome shotgun sequence
assemblies [19, 20].
As compared to dealing with the whole genome, chromosome
sorting offers a massive and lossless reduction of DNA sample com-
plexity, and this both simplifies the analysis of DNA sequence data
and reduces project costs. However, there is an additional group of
applications where sorting all chromosomes of the chromosome com-
plement is beneficial. Since the purified sample consists only of
mitotic chromosomes, this provides the opportunity to characterize
the proteome of mitotic metaphase chromosomes and to reveal spa-
tial organization of chromosomal DNA using the chromosome confor-
mation capture methods. In the following, we describe chromosome
analysis and sorting in higher plants, pointing to critical steps and out-
lining the methods for selected applications.
2 | SAMPLE PREPARATION
2.1 | Choice of plant material
A majority of cells forming a plant organism are in interphase. Cells
in the metaphase stage of the cell cycle, which is the only stage at
which condensed chromosomes are physically separated from each
other, are rare or completely absent. In order to increase their fre-
quency and accumulate cells at metaphase, cells must be induced
to cycle and divide. In principle, different plant parts, organs, and
cell suspensions may be used, and a number of attempts have been
made to verify their suitability. Suspension cultured cells [21–23]
initially seemed an ideal system, since cells grown in vitro under
controlled conditions may be easily treated with chemical agents
in order to induce mitotic synchrony. However, cell cultures can-
not be easily established in every species [24], and they are often
genetically heterogeneous [22] and karyologically unstable [25]. A
majority of cells in differentiated tissues in vivo, such as leaf meso-
phyll are arrested at G1 phase of cell cycle. As all cells are initially
at the same cell cycle stage, they should traverse S and M phases
synchronously upon cell cycle induction. Unfortunately, the exper-
iments by Conia et al. [26] did not confirm this expectation and
mitotic synchrony observed after transferring isolated mesophyll
protoplasts to a nutrient medium was low.
Buds and root tips belong to a few plant organs comprising prolif-
erating cell populations, with root tips having large populations of
cycling cells that are easy to manipulate [27, 28]. Seeds are available
for many plant species, and young seedlings can be grown hydroponi-
cally and their roots treated with chemical agents to achieve high pro-
portions of metaphase cells [27, 29]. An important advantage is that
the meristem cells are karyologically stable. If a certain genotype can
only be propagated vegetatively, actively-growing roots may be
obtained from genetically transformed “hairy” root cultures [30, 31].
It is also possible to grow vegetative organs, such as bulbs or stem
cuttings, using hydroponic systems to initiate root formation and
growth [32, 33]. Due to these advantages, almost all experiments on
chromosome analysis and sorting in plants reported so far have uti-
lized samples prepared from synchronized root tip meristems [34–36].
The subsequent parts of this paper will deal with this type of source
tissue.
2.2 | Cell cycle synchronization and accumulation
of cells in metaphase
In order to enable treatments with various compounds to achieve high
frequency of cells at metaphase, it is recommended to grow seedlings
in a hydroponic system (Figure 1). The nutrient medium should have a
defined composition and Hoagland's solution [37] has been generally
used [27]. Its concentration should be adjusted so that it does not
negatively affect mitotic activity in root tip meristems. As cell cycle
F I G U R E 1 Plastic trays with open-mesh baskets supporting
young seedlings grown in hydroponic. Note that the Hoagland's
nutrient solution is aerated using aquarium bubble air stones to avoid
hypoxia. Photo Pavlína Jáchimová, ASCR
DOLEŽEL ET AL. 3

kinetics is sensitive to temperature, all treatments in such system are
performed at constant temperature in a biological incubator, the solu-
tions are pre-heated to the required temperature, and are also aerated
to ensure an adequate oxygen supply.
Root tips of young seedlings are first treated with a DNA syn-
thesis inhibitor to accumulate a majority of cycling population at
G1-S interface. Hydroxyurea, a compound which inhibits ribonu-
cleotide reductase [38], has been by far the most frequently used
because of its effectiveness and low cost. After the cycling cells
are accumulated at the G1-S interface, the seedlings are trans-
ferred to inhibitor-free nutrient medium to allow the cells to
resume the cell cycle and to traverse synchronously through S and
G2 phases. Cytological evaluation of the proportion of mitotic cells
at different time points following the release from the block, helps
to identify the recovery time at which the highest proportion of
cells reaches mitosis. In addition, flow cytometric analysis of DNA
content of meristem tip nuclei makes it possible to follow the pro-
gression through the cell cycle of the synchronized population
after the release of the block (Figure 2). The results identify the
optimal concentration of DNA synthesis inhibitor and the treat-
ment duration to accumulate a majority of cells at the G1-S inter-
face and yet allow a rapid transit through S and G2 after the
release from the block [27]. The treatments optimized for a range
of plant species are listed in Table 1. In fact, it has been found that
the highest degree of mitotic synchrony is achieved at hydroxy-
urea concentrations that allow the cells arrested at G1-S interface
to escape the block a few hours before the roots are transferred to
hydroxyurea-free solution (Figure 2) [27].
In order to accumulate mitotic cells at metaphase, seedlings are
transferred to a medium supplemented with mitotic spindle inhibitor.
From a range of compounds with this property, only a few synthetic
herbicides have been used, the most popular being amiprophos-
methyl, followed by oryzalin and trifluralin [29, 55, 56]. Their advan-
tage over the traditionally used colchicine is that they are effective in
lower concentrations, are less toxic to plant cells and are easier to
manipulate. It is recommended that the compounds be used at the
lowest concentration that arrests chromosomes at metaphase and
that the treatment time is kept as short as possible (e.g., limited to
about 2 h) to avoid separation of sister chromatids (Table 1). The opti-
mum timing of the metaphase block to achieve the highest frequency
of cells at metaphase is determined cytologically. In some species,
including maize and rapeseed, the chemicals inhibiting mitotic spindle
may induce chromosome clumping. In maize, this can be avoided by
replacing common spindle inhibitors by a treatment with nitrous oxide
[57]. In some species, spreading of metaphase chromosomes across
the cell volume can be improved by an ice-water treatment [33].
Seedlings with synchronized roots may be treated with ice-water
overnight, allowing convenient scheduling of the next step of the pro-
tocol for the following morning. If the overnight treatment is not part
of the protocol, root tips must be fixed immediately after the meta-
phase block to avoid separation of sister chromatids and chromosome
decondensation.

F I G U R E 2 Cell cycle synchrony in root tip cells of barley (Hordeum vulgare) cv. Morex induced by a treatment with 2.0 mM hydroxyurea for
18 h. cell nuclei were isolated from root tips at different time points after a transfer to hydroxyurea-free nutrient medium and their DNA content
was estimated by flow cytometry. The upper left histogram shows DNA content distribution in non-treated root tips. The sample taken at 0 h
shows a large fraction of nuclei in S phase, indicating a precocious release from the hydroxyurea block. A majority of cells are at G2 phase at 6 h
when they start entering mitosis
4 DOLEŽEL ET AL.

T A B L E 1 Plant species in which chromosome suspensions used for flow cytometric analysis and sorting were prepared by mechanical
homogenization of formaldehyde-fixed root tips

Recovery
Hydroxyurea time Metaphase accumulation

Concentration Duration Duration Chemical agent and Treatment References (only the first
Species (mM) (h) (h) concentration duration (h) reports are cited)
Aegilops spp. (biuncialis, comosa, 1.25 18 5 APM 2.5 μM 2 + ice [39]
geniculata, umbellulata) overnight
Aegilops spp. (cylindrica, 1.25 18 5 APM 2.5 μM 2 + ice [40]
markgrafii, triuncialis) overnight
Aegilops spp. (speltoides, 1.25 18 5 APM 2.5 μM 2 + ice [41]
tauschii) overnight
Agropyron cristatum 1.25 18 4.5 APM 2.5 μM 2 + ice [42]
overnight
Arachis hypogea 3.0 18 5 APM 5 μM 2 [33]
Asparagus officinalis 3.0 18 6 APM 5 μM 2 [43]
Avena sativa 2,0 18 4,5 Oryzalin 10 μM 5 [33]
Avena strigosa 2.0 18 5 Oryzalin 5 μM 2 Unpublished
Cicer arietinum 1.25 18 4 Oryzalin 5 uM 2 + ice [44]
overnight
Crocus sativus 0.5 18 5 APM 5 μM 3 + ice [33]
overnight
Dasypyrum villosum (syn. 1.25 18 5 APM 2.5 μM 2 + ice [45]
Haynaldia villosa) overnight
Elymus elongatum 1.25 18 5 APM 2.5 μM 2 + ice [33]
overnight
Festuca pratensis 1.0 18 4.5 N2O (506.625 kPa) 2 [46]
Gossypium hirsutum 4.5 18 3.5 Oryzalin 10 μM 2 + ice [33]
overnight
Hordeum vulgare 2.0 18 6.5 APM 2.5 μM 2 + ice [47]
overnight
Hordeum vulgare cv. Barke 1.5 18 5.5 APM 2.5 μM 2 + ice [33]
overnight
Lens culinaris 1.25 18 4.5 APM 10 μM 2 + ice [33]
overnight
Lolium perenne 1.5 18 4.5 APM 5 μM 2 [33]
Lupinus angustifolius 3.0 18 4 Oryzalin 2.5 μM 2 + ice [33]
overnight
Medicago sativa 4.5 18 6 APM 2.5 μM 3 [33]
Pisum sativum 1.25 18 4.5 APM 10 μM 2 + ice [48]
overnight
Rumex acetosa 2.0 18 5 Oryzalin 10 μM 2 + ice [33]
overnight
Saccarum officinarum 3.5 18 1.0 APM 2.5 μM 3 [32]
Secale cereale 2.5 18 6.5 APM 10 μM 2 + ice [49]
overnight
Silene spp. (latifolia, dioica) 2.0 18 5 oryzalin 2.5 μM 2 [33]
Triticum aestivum 2.0 18 5.5 APM 2.5 μM 2 + ice [33]
overnight
Triticum monococcum 1.25 18 5 APM 2.5 μM 2 + ice [50]
overnight
Triticum militinae 2.0 18 5.5 APM 2.5 μM 2 + ice [51]
overnight
Triticum dicoccoides 2.0 18 5.5 APM 2.5 μM [52]
DOLEŽEL ET AL. 5

TABLE 1 (Continued)

Recovery
Hydroxyurea time Metaphase accumulation

Concentration Duration Duration Chemical agent and Treatment References (only the first
Species (mM) (h) (h) concentration duration (h) reports are cited)
2 + ice
overnight

Triticum durum 1.25 18 5 APM 2.5 μM 2 + ice [53]

overnight
Triticum timopheevii 2.0 18 5.5 APM 2.5 μM 2 + ice Unpublished
overnight
Triticum urartu 2.0 18 5.5 APM 2.5 μM 2 + ice [41]
overnight
Vicia faba 1.25 18.5 4.5 APM 2.5 μM 2 [29]
Vicia sativa 2.5 18.5 3.5 Oryzalin 5 uM 2 [54]
Vigna unguiculata 1.25 18 4 Oryzalin 2.5 μM 2 + ice [33]
overnight
Zea mays 3.0 18 3.5 N2O 506.625 kPa 3 [33]

Abbreviation: APM, amiprophos-methyl.

2.3 | Release of intact mitotic chromosomes from
cells
All protocols on the preparation of chromosome suspensions from
synchronized root tip meristems published to date have been based
on mechanical homogenization of root tips. Doležel et al. [29] showed
that a mild fixation with formaldehyde prior to root tip chopping
increases the yield of intact chromosomes. In the original protocol
[29], root tips of Vicia faba were homogenized by chopping using a
scalpel in a chromosome isolation buffer, and chromosome clumps
were dispersed by syringing through a hypodermic needle. Although
this procedure results in high quality chromosome suspensions, it is
not practical for species with small roots and for experiments which
require many samples. Thus, chromosome suspensions are almost uni-
versally prepared by homogenization of root tips using a mechanical
homogenizer as described by Gualberti et al. [48]. Nevertheless, chop-
ping can still be useful when handling delicate and/or rare samples.
In both versions of the protocol, formaldehyde fixation of root
tips enhances the resistance of metaphase chromosomes to mechani-
cal shearing during the isolation and subsequent flow cytometric anal-
ysis and sorting. To achieve the highest yield of intact chromosomes,
the extent of the fixation, which is defined by formaldehyde concen-
tration and length of the fixation, needs to be determined experimen-
tally for each species by microscopic observation of crude
chromosome suspensions and flow cytometric analysis of chromo-
some samples [29]. The observations of Lee et al. [56] indicate that is
possible to avoid the fixation step, however, at the expense of a much
lower chromosome yield.
The chemical composition of chromosome isolation buffer is criti-
cal to ensure high chromosome yields and stabilize their morphology.
The composition and pH must also be compatible with chromosome
staining conditions, and depending on the use of sorted chromosomes
in downstream applications, the buffer must protect DNA and pro-
teins from degradation. The polyamine-based LB01 buffer [58],
proved to satisfy these requirements. However, it needs to be modi-
fied if high molecular weight (HMW) DNA is to be prepared from
flow-sorted chromosomes [59], or if chromosomal proteins are iso-
lated for proteomic analyses [60].
In general, isolated plant chromosomes are not stable over time, and
therefore should be analyzed on the day of isolation. Stability can be
species-specific: for example, our unpublished results indicate that chro-
mosome suspensions prepared from formaldehyde-fixed roots of Vicia
faba and Pisum sativum can be stored at 4 C for several months without
detectable negative effects. On the other hand, cereal chromosomes
deteriorate more rapidly and even 1 day-old chromosome samples display
lower resolution of chromosome peaks or populations on flow karyo-
types. It is worth mentioning that isolated wheat chromosomes denatured
by sodium hydroxide, as done in the fluorescence in situ hybridization in
suspension (FISHIS) protocol [61] are suitable for flow karyotyping for as
long as several months. Moreover, the addition of hexylene glycol
improved the long-term stability of Vicia faba chromosome suspensions
and avoided chromosome clumping during storage [62].
3 | CHROMOSOME ANALYSIS (FLOW
KARYOTYPING)
3.1 | Choice of instruments
Chromosome samples may be analyzed on any flow cytometer with
optical setup suitable for the quantification of fluorochrome(s) used to
label the isolated chromosomes. The ability to analyze light scatter is
helpful, but the main threshold is settled on DNA fluorescence chan-
nel. The instrument must be stably aligned to achieve the highest
6 DOLEŽEL ET AL.

possible resolution during long runs. If the instrument has inter-
changeable flow chambers/nozzles, small orifices (typically 70 μm in
diameter) are recommended as this results in higher resolution and
chromosome discrimination. A simple sheath fluid, comprising 50 mM
NaCl, is suitable for all chromosome analysis and sorting experiments
(unpublished).
Prior to the analysis, chromosome samples are filtered through a
nylon mesh (21–36 μm pore size) to eliminate larger particles. Flow
cytometric chromosome analysis, or flow karyotyping, leads to chro-
mosome classification according to their optical parameters. The most
important has been fluorescence of dyes bound to DNA, and all fluo-
rescence pulse parameters, including pulse height (FL-H), area (FL-A)
and width (FL-W) are informative [63, 64]. In addition, light scattering
properties are useful to gate out debris present in chromosome sam-
ples. Due to the poorer light collection properties of the first
generation jet-in-air flow cytometers and sorters, lasers with output
power of several hundred milliwatt were needed to achieve the
required sensitivity and resolution of fluorescence signals [62]. The
newer generation of flow cytometers in which optical parameters are
interrogated in an enclosed stream allows high resolution flow
karyotyping with air-cooled compact solid state lasers output power
of as low as 20 mW [64] (Figure 3).
3.2 | Univariate flow karyotyping
In single parameter, or univariate, flow karyotyping, chromosomes are
classified according to fluorescence of a single fluorochrome bound to
DNA, which is the most important signal in flow cytometric chromosome
analysis. In addition, light scatter properties are analyzed and forward light

F I G U R E 3 Comparison of bivariate flow karyotyping (GAA-FITC vs. DAPI) by two flow cytometers differing in optical design and laser output
power. (A, B, C) BC Cytoflex S equipped with solid state low power lasers (375 nm at 20 mW and 561 nm at 30 mW). (D) BD FACSVantage SE jet
in air sorter equipped with two water-cooled argon lasers (357–361 nm at 200 mW and 488 nm at 400 mW). Chromosomes isolated from durum
wheat (Tricum durum, 2n = 4x = 28) cv. Cappelli were labeled by FISHIS using the same probe GAA7 and counterstained by DAPI. The
chromosome discriminative capability of the bivariate analysis is shown by single chromosome regions (in color)
DOLEŽEL ET AL. 7

scatter (FSC-A) is a recommended parameter to couple with DNA fluores-
cence pulses (FL-A) to discriminate small debris and chromosomes
(Figure 4(A)). The analysis of chromosome fluorescence intensity (FL-A)
results in histogram of relative fluorescence intensity, representing the
univariate flow karyotype (Figure 4(B)). The ability to discriminate individ-
ual chromosomes within the flow karyotype depends on differences in
chromosome DNA content, or DNA base content, among the chromo-
somes within karyotype and on the resolution of the analysis. The latter
depends on: (i) the quality of the chromosome sample, which should con-
tain the highest proportion of intact chromosomes and lowest of dam-
aged chromosomes and chromosome clumps; (ii) the method used to
stain chromosomal DNA; and (iii) the technical parameters of the instru-
ment and quality of its alignment.
From the range of DNA binding fluorochromes that are available,
only a few have been used for univariate flow karyotyping in plants.
These included the DNA intercalators ethidium bromide [21] and
propidium iodide [22], the fluorescent antibiotics mithramycin and
chromomycin A3 [22], and DAPI, which binds preferentially to AT-rich
regions of DNA [29]. The choice of a fluorochrome depends primarily
on the available lasers and the optical setup of the instrument. Given
the lack of comparative analyses, it seems that for native non-fixed
chromosomes, any of the above-mentioned fluorochromes are useful.
The situation is different for chromosomes isolated after formalde-
hyde fixation. Here, only DAPI is recommended as its binding is not
affected by the fixation [62], most probably a consequence of the
way DAPI binds to chromatin [65, 66].
3.3 | Bivariate flow karyotyping
In a majority of plant species analyzed so far, differences in relative
DNA content, or DNA base content among the chromosomes, are too
small to permit discrimination of all chromosomes using a single DNA
fluorochrome. Unfortunately, bivariate flow karyotyping after simulta-
neous chromosome staining with AT- and GC-binding fluorochromes,
which is a standard in human flow karyotyping [67], does not lead to a
significant improvement [68, 69] and has been rarely used. An alterna-
tive, and to date the only successful method for bivariate flow
karyotyping in plants, has been to fluorescently label a repetitive DNA
sequence which is abundant and non-homogenously distributed
across the chromosomes of the karyotype.
First attempts to label particular DNA sequences of chromosomes
in suspension by modified primed in situ DNA labeling (PRINS) [70]
brought some promising results [71], but the method suffered from
poor reproducibility and has not been used further. Afterwards,
attempts to apply fluorescence in situ hybridization (FISH) to chromo-
somes in suspension were made and although Ma et al. [72] achieved
chromosome labelling in suspension, the suitability of the samples for
flow cytometry was not confirmed. The difficulties were finally over-
come by Giorgi et al. [61], who developed a protocol for FISHIS, which
is fully reproducible (Figure 4(C)). Their method differs from standard
FISH protocols in that chromosomal DNA is denatured by alkali and
no washing steps are performed, avoiding chromosome clumping. To
date, the method has been used to label different microsatellite
repeats on chromosomes in suspension from several plant species
(Table 2). The probes are mainly fluorescent oligonucleotides, which
do not require DNA denaturation prior to use. In some species, chro-
mosome resolution can be further improved by using oligonucleotide
probes for two different microsatellites [50, 73, 74]. Lucretti et al. [74]
demonstrated that other repetitive sequences, such as the 18S-5.8S-
26S ribosomal DNA [77] labeled by a fluorochrome by nick-transla-
tion, can also be used as probes for FISHIS. It is important that FISHIS
can be easily integrated into chromosome sample preparation pipeline
as it can be performed at room temperature in less than 2 h.

F I G U R E 4 Flow cytometric chromosome analysis (flow karyotyping) in bread wheat (Triticum aestivum, 2n = 6x = 42) cv. Chinese spring.
(A) Dot-plot DAPI-A versus FSC-A is used to select the population of intact chromosomes and exclude small chromosome fragments and cell
debris (gate window is shown as black rectangle). (B) Monovariate flow karyotype (DAPI-A) shows three composite peaks representing groups of
chromosomes; only chromosome 3B is represented by a well discriminated peak visible on the right side of the distribution. (C) Bivariate flow
karyotype GAA-FITC versus DAPI-A permits discrimination of almost all 21 chromosomes of bread wheat. Note that in panel (C) the Y axis is log
scale to accommodate the large range of FITC fluorescence intensities after labelling GAA microsatellites by FISHIS
8 DOLEŽEL ET AL.

T A B L E 2 Plant species for which chromosome labeling by FISHIS 4 | CHROMOSOME SORTING

has been used for flow cytometric chromosome sorting

Species Probe Reference 4.1 | Chromosome population gating and sort

Aegilops biuncialis (GAA)7 Unpublished windows
Aegilops comosa (GAA)7 + (ACG)7 [73]
There are two basic applications of chromosome sorting. The first is
Aegilops geniculata (GAA)7 Unpublished
the purification of mitotic chromosomes in bulk to study their molecu-
Aegilops markgrafii (GAA)7 + (ACG)7 [73]
lar organization [83, 84], prepare microscopic slides for mapping DNA
Aegilops sharonensis (GAA)7 Unpublished
sequences by FISH at high spatial resolution [85, 86], and localize
Aegilops speltoides (GAA)7 + (ACG)7 [73]
chromosomal proteins by immunocytochemical methods [87, 88]. The
Aegilops triuncialis (GAA)7 Unpublished
second, and by far more frequent application, has been the purifica-
Aegilops umbellulata (GAA)7 [73] tion of particular chromosomes to dissect nuclear genomes to chro-
Aegilops uniaristata (GAA)7 Unpublished mosomes, or small groups of chromosomes, to aid in genome
Agropyron cristatum (GAA)7 [42] sequencing [16–18], targeted development of DNA markers [8–11],
Dasypirum villosum (H. villosa) (GAA)7 [61] and gene cloning [14, 15, 89–92].
Dasypyrum villosum (H. villosa) pTa71 (rDNA) [74] To discriminate a particular population of chromosomes from other
Triticum aestivum (GAA)7 (AG)12 [61] particles in the sample, optical parameters which define the population of

Triticum aestivum (GAA)7 [75] interest are selected. In monovariate flow karyotyping, it is advisable first
to electronically filter out small debris by setting a gate on a dot-plot
Triticum aestivum (transgenic (GAA)7 [76]
lines) FSC-A versus DAPI FL-A, and then define the region of interest on a dot-
Triticum. dicoccoides (GAA)7 [52] plot DAPI FL-A versus DAPI FL-W (Figure 5(A)). Setting the sort windows

Triticum durum (AG)12 (AAT)7 [61] may be easier on bivariate flow karyotypes, such as DAPI FL-A versus
(AAC)5 GAA-FITC FL-H, where the chromosome populations can be more cleanly
Triticum durum (AG)12 [74] separated (Figure 5(B)). The definition of sort windows is critical since

Triticum militinae (GAA)7 [51] these determine the chromosomes that are sorted and affect the degree
of contamination of the sorted fractions by other chromosomes, chromo-
Triticum monococcum (ACC)5 + (GAA)7 [50]
some clumps and fragments.
Triticum timopheevii (GAA)7 Unpublished

4.2 | Discrimination and sorting chromosomes of

3.4 | Flow karyotype description interest

Interpretation of flow karyotypes requires the assignment of chromo-
somes to individual peaks on histograms of fluorescence intensity, or
in populations of bivariate flow karyotypes. This is a difficult task if
the analysis is done using an instrument without a sorting option. In
principle, one may compare the experimental data with theoretical
models calculated based on relative chromosome lengths [78, 79] or
even estimating the total fluorescence labeling intensities coupled
with DNA content [80]. However, this is a rough approximation and
differences in AT/GC content, and heterogeneity in chromatin con-
densation among chromosomes in a karyotype may cause differences
between theoretical and experimental flow karyotypes [81]. The task
is somewhat easier if one or more chromosomes differ significantly in
size, or AT/GC content from other chromosomes. Such situation may
be observed in chromosome deletion, translocation, or alien addition
lines [62]. However, the only truly reliable way to assign chromo-
somes to peaks or populations on a flow karyotype is to sort chromo-
somes and identify them after microscopic observation, or using PCR
with specific primers, or by sequencing. Microscopic observation is
the preferred method, since it allows a detailed characterization of the
sorted fraction by determining the presence of individual chromo-
some types [82].
The karyotypes of some plant species comprise two or more chromo-
somes that are similar in size and DNA repeat content, and currently
available methods for fluorescent staining of chromosomal DNA or
labeling particular DNA repeats may fail to discriminate between
these chromosomes. The consequence is the appearance of compos-
ite peaks on monovariate flow karyotypes, or mixed populations on
bivariate flow karyotypes. The extent of the problem varies between
species, from the ability to discriminate all chromosomes using a single
FISHIS probe, such as in Haynaldia villosa [61] and some Aegilops spe-
cies [73] (see Table 2 for the list of probes used for FISHIS), to the
ability to discriminate only a few chromosomes even after FISHIS
labeling, such as in Secale cereale. There is a clear effect of polymor-
phisms in DNA repeat content, and whereas in some Hordeum vulgare
cultivars only five of the seven chromosomes can be resolved, all
seven chromosomes can be distinguished in other cultivars.
In polyploids, and in bread wheat in particular, one solution has
been to use lines with chromosome deletions, such as the telocentric
stocks [3]. In polyploids, it is also possible to develop alien chromo-
some addition lines (Figure 6). Thus, wheat-rye chromosome addition
lines were used to purify rye chromosomes for shotgun sequencing
and draft genome assembly [16], and wheat-barley telosome addition
DOLEŽEL ET AL. 9

F I G U R E 5 Setting sort windows (red rectangles) to isolate particular chromosomes from bread wheat (Triticum aestivum, 2n = 6x = 42)
cv. Chinese Spring. (A) in monovariate flow karyotyping, when chromosomes are stained just by DAPI, sort window is set on a dot-plot DAPI-A
versus DAPI-W. The example is given for sorting the largest chromosome 3B. (B) Bivariate flow karyotyping DAPI-A versus GAA-FITC allows
discrimination of chromosomes that cannot be discriminated using the monovariate analysis. The window is set to sort chromosome 5B, which is
included in the large composite peak representing 10 different chromosomes on a monovariate flow karyotype

F I G U R E 6 Chromosome discrimination in bread wheat (Triticum aestivum, 2n = 6x = 42) cv. Chinese Spring with a wild-type karyotype (left
column), wheat-barley 7HS ditelosomic addition line (middle column) and wheat chromosome translocation line T5BS.7BS + T5BL.7BL (right
column). The upper row shows monovariate flow karyotypes obtained after the analysis of DAPI fluorescence pulse area (DAPI-A) and red arrows
point to peaks representing chromosomes, which can be easily discriminated. The lower row shows bivariate flow karyotypes DAPI-A versus
GAA-FITC and red rectangles define the populations of chromosomes represented by peaks in monovariate flow karyotypes (upper row). Note
that the small translocation chromosome T5BS.7BS is included in the composite peak of wild-type chromosomes 1D, 4D, and 6D on a
monovariate flow karyotype, while it is clearly discriminated in the bivariate flow karyotype
10 DOLEŽEL ET AL.

lines facilitated isolation of barley chromosome arms for SNP mapping
using DNA arrays [93] and sequencing to obtain a high resolution
sequence-based gene map of the crop [17]. Certain translocations
may alter chromosome size, and, as they may be induced also in dip-
loids, provide an attractive way to discriminate particular chromo-
somes [47, 62, 63, 94]. However, the development of such lines is
time-consuming, and they cannot be easily obtained. Although the
sorted translocation chromosomes facilitate gene mapping [47,
94, 95], their DNA may not be directly suitable for other purposes,
such has the genome sequence assembly.
If the chromosome of interest cannot be distinguished by any of
the above-mentioned strategies, the option is to sort single copies of
chromosomes from a composite peak/population into a set of PCR
tubes, or wells in a microtitre plate, and amplify DNA of single chro-
mosomes to produce microgram amounts of DNA [96]. As anonymous
chromosomes are sorted, their identity is revealed only after amplifi-
cation using specific primers or shotgun sequencing. Although the
spectrum of applications may be limited due to variable DNA frag-
ment lengths, ranging from 300 bp to 20 kb, and by amplification that
is not fully representative [96], the approach has been used recently
to validate reference genome assembly of garden pea and reveal chro-
mosome translocations in its wild relatives [19].
a microscopic slide into a small drop of chromosome isolation buffer
LB01 supplemented with 5% (w/v) sucrose to preserve chromosome
morphology during air-drying [81]. The slides prepared this way are
used for FISH with probes that enable identification of individual
chromosomes within a karyotype (Figure 7). The added bonus of
labelling chromosomes by FISHIS prior to sorting is that the chromo-
somes sorted onto a microscope slide do not have to be labeled by
FISH and are directly suitable for fluorescence microscopy as the fluo-
rescent labels are not removed during the sorting. Evaluation of a few
hundred chromosomes is sufficient to characterize the population of
sorted chromosomes.
This approach is feasible only with droplet sorters as the volume
droplets in which the particles are sorted is small (e.g., 1 nl) and chro-
mosomes are spread across a small area on the slide making micro-
scopic observation easier. In principle, fluidic switch sorters could also
be used. However, as the volume of the sorted fraction is much larger,
it is necessary to pellet the chromosomes before transferring them
onto a slide, and the centrifugation can result in chromosome loss and
clumping.
4.4 | The yield of sorted chromosomes

As in other applications of flow cytometric sorting, chromosome sort

4.3 | The identity of sorted chromosomes and the
purity in sorted fractions
The observation of sorted fractions by fluorescence microscopy is
strongly recommended to assign chromosomes to populations within
a flow karyotype, confirming that a chromosome of interest is sorted,
and to identify other chromosomes or fragments that contaminate the
sorted fraction. To do this, about 1000 chromosomes are sorted onto
purity and yield are inversely proportional, and setting up a very tight
sort window may lead to higher purity, but also low sort rates. It is
thus critical to run chromosome samples at rates securing high resolu-
tion of DNA peaks/chromosome populations. When analyzing chro-
mosome samples prepared from bread wheat according to Vrána et al.
[81], the recommend sample rate is about 2000 particles/s. The exact
size, shape, and position of the sort window needs to be determined
experimentally, relying on the results of microscopic observation of

F I G U R E 7 Images of chromosomes flow-sorted onto microscope slides. (A) Chromosomes 5B sorted from bread wheat (Triticum aestivum)
cv. Chinese spring. (B) Chromosomes 1B and 6B bearing NOR loci sorted from durum wheat (Triticum durum) cv. Creso. Prior to analysis and
sorting, chromosomes in suspension were labeled by FISHIS: (A) using a probe (GAA)7-Alexa488 (yellow–green), and (B) using a probe for
pTa71-Cy3 (red). Note that during landing on a slide, a satellite has been lost from the chromosome on the top left side. In both cases,
chromosomes were stained by DAPI (blue). The specific fluorescent labeling patterns obtained after FISHIS remain stable during the sorting and
facilitate identification of the sorted chromosomes. Bars = 10 μm
DOLEŽEL ET AL.
flow-sorted particles. In a typical experiment, a chromosome of bread
wheat can be sorted from a sample prepared according to Vrána et al.
[81] at rates of 5–10 particles/s. In order to keep the final volume of
the sorted fraction low, the sorting is done using the single-
droplet mode.
5 | C HR OM OSOM E SO R TI N G F O R
VARIOUS APPLICATIONS
Plant chromosomes sorted by flow cytometry have been used in a
variety of applications, including molecular cytogenetics, molecular
biology, genome sequencing, and gene cloning, and proteomics [36].
The way chromosomes are sorted for each application may differ as
outlined below.
5.1 | Preparation of high molecular weight DNA
for BAC library construction, optical mapping and
long-read DNA sequencing
In order to maintain chromosomal DNA integrity, chromosomes
should be isolated into a buffer optimized for this purpose [59]. As
these applications require hundreds of nanograms to several micro-
grams of DNA, a large number of chromosomes (typically 105–106)
must be sorted. When determining the number of chromosomes to be
sorted, one needs to consider the molecular size of the sorted chro-
mosome, the fact the mitotic chromosomes have two chromatids, and
also the losses during the preparation of HMW DNA from sorted
chromosomes, which may reach 75%–80%. In bread wheat, a typical
sort yield is 400,000–900,000 chromosomes per working day, hence
the preparation of HMW DNA takes days or even weeks, and many
chromosome samples will need to be prepared. The chromosomes are
sorted into 1.5-ml DNA low-binding polystyrene tubes in batches,
ranging from 200,000 to 700,000 for bacterial artifical chromosome
(BAC) library construction and optical mapping, respectively. It is rec-
ommended to sort into 1.5× chromosome isolation buffer (220 μl for
BAC library construction and 770 μl for optical mapping) to counter-
balance dilution by sheath fluid.
5.2 | Sequencing amplified chromosomal DNA
Many applications in molecular biology and genomics do not require
high molecular weight DNA, and DNA samples with fragments sizes
up to 20 kb are sufficient. This offers the possibility to employ whole
genome amplification of DNA to produce microgram amounts of DNA
from a smaller number of chromosomes [97]. The number of chromo-
somes that need to be sorted depends on chromosome size, being
determined such that the equivalent of 40 ng sorted DNA is obtained.
In case of bread wheat, this corresponds to about 20–30 thousand
chromosomes, depending on chromosome size. The chromosomes are
sorted into 0.5-ml PCR tubes containing 40 μl sterile deionized water.
11
The tubes with chromosomes are spun down using a microcentrifuge
immediately after sorting and kept at −20 C until used for DNA
amplification.
5.3 | Sequencing non-amplified chromosomal DNA
The introduction of protocols for Illumina sequencing libraries requir-
ing nanogram amounts of DNA or less, made it possible to skip the
chromosome amplification step. In fact, sequencing non-amplified
chromosomal DNA results in better assemblies compared to those
from amplified DNA [90]. The number of sorted chromosomes and
the way they are collected is the same as in the protocol for sequenc-
ing amplified DNA. The tubes with chromosomes are spun down using
a microcentrifuge immediately after sorting and kept at −20 C until
used for preparation of DNA sequencing libraries.
5.4 | Single copy chromosome DNA sequencing
As minute amounts of DNA are amplified in this case, special precau-
tions need to be taken to avoid sample contamination by DNA of
other organisms and the samples should be processed under sterile
conditions in a laminar flow hood. Single chromosomes are sorted into
3 μl whole genome amplification mix in 0.2 ml PCR tubes, which are
sterilized by autoclaving [96]. To prepare a positive control, 1000
chromosomes are sorted into another tube. A negative control is the
0.2 ml PCR tube with the amplification mix and without chromo-
somes. All tubes with chromosomes should be spun down using a
microcentrifuge immediately after sorting.
5.5 | Chromosome conformation capture (Hi-C)
Chromosome flow-sorting can be used to study chromatin conforma-
tion by inferring its spatial contacts in all-versus-all manner using a
chromosome conformation capture method termed Hi-C [98]. As the
formaldehyde fixation is a crucial step in this analysis and directly
impacts sequencing results, it needs to be optimized and usually
slightly differs from the standard protocols used to prepare suspen-
sions of intact chromosomes. In order to obtain sufficient number of
contacts between DNA loci, 3–5 million chromosomes per replicate
are sorted into 15 ml protein low-binding tubes with 2 ml LB01
buffer, which are kept on ice during the entire sorting procedure.
5.6 | Proteomic analysis
Proteomic analysis requires that proteins of flow-sorted chromosomes
are protected from degradation. This is achieved by using a modified
chromosome isolation buffer and a modified buffer into which the
chromosomes are sorted. In order to improve the accessibility of chro-
mosomal proteins, it is also recommended to reduce the extent of
12
formaldehyde fixation of synchronized root tips. This application has
been developed to study protein composition of mitotic metaphase
chromosomes [60] and all chromosomes of a plant are sorted,
avoiding the need to discriminate particular chromosome type. The
sample rate may thus be higher, reaching 6000 chromosomes/s, and
sort yields range between 3.5 and 6.6 million chromosomes/working
day. In order to isolate enough proteins for mass spectrometry, 5–10
million sorted chromosomes are required. To accommodate the high
number of droplets with sorted chromosomes, sorting is done into
5 ml polystyrene tubes, containing 1 ml of modified chromosome iso-
lation buffer.
6 | B ES T P R A CT I CE S
• The most suitable material for preparing liquid chromosome sus-
pensions are meristem tips of actively growing roots of young
seedlings, bulbs, or stem sections.
• When setting-up a methodology for a new species, sufficient
amount of plant material (seeds, bulbs, or stem sections) should be
available to allow for a thorough optimization of individual steps of
the protocol.
• To prepare liquid chromosome suspensions suitable for flow cyto-
metry, synchronized root tips should contain at least 40% of cells
accumulated at mitotic metaphase.
• The use of DNA low-binding tubes is recommended for sample
preparation and storage.
• Mild fixation of plant material by formaldehyde prior to mechanical
homogenization increases chromosome yields, preserves chromo-
some morphology and improves their mechanical stability.
• To stain DNA of chromosomes isolated after formaldehyde fixa-
tion, only DAPI is recommended and the instrument should be
equipped with UV, near-UV, or violet laser for optimal DAPI
excitation.
• The flow sorter must be precisely aligned to achieve high resolu-
tion in DNA fluorochrome channel(s) with the coefficient of varia-
tion of chromosome peaks as low as possible (ideally less than 2%).
The instrument alignment must be stable during sort runs.
• The chromosome content of populations identified by flow
karyotyping and the purity at which they can be flow-sorted should
be checked by microscopic observation of the populations sorted
onto a microscopic slide. The check should be repeated regularly
during long sort runs.
• Bivariate flow karyotyping DAPI fluorescence versus fluorescence
of FISHIS-labeled DNA repeat(s) should always be tested to verify
if it helps to improve the discrimination of chromosome
populations.
• DNA probes suitable for FISHIS need to be selected empirically for
each new species. In order to achieve a specific and intensive
FISHIS labelling, pH and time of the denaturation step as well as
final pH of the liquid chromosome suspension after stopping the
denaturation must be carefully controlled.
DOLEŽEL ET AL.
ACKNOWLEDG MENTS
Funding was provided in part by grants from the European Regional
Development Fund project “Plants as a Tool for Sustainable Global
Development” (CZ.02.1.01/0.0/0.0/16_019/0000827 to J.D.,
P.C. and I.M.) and the Italian Ministry of Agriculture “Progetto MAPPA
5A” (D.M. 7398/7303/08 to S.L. and D.G.).
CONFLIC T OF INT ER E ST
The authors declared no potential conflict of interest.
AUTHOR CONTRIBU TIONS
Jaroslav Doležel: Conceptualization; funding acquisition; supervision;
writing-original draft; writing-review & editing. Sergio Lucretti: Con-
ceptualization; funding acquisition; project administration; writing-
original draft; writing-review & editing. István Molnár: Visualization;
writing-review & editing. Petr Cápal: Visualization; writing-review &
editing. Debora Giorgi: Visualization; writing-original draft; writing-
review & editing.
OR CID
Jaroslav Doležel https://orcid.org/0000-0002-6263-0492
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